Higher order organization of chromatin generates structures that are essential for high fidelity chromosome segregation, DNA damage repair, and the regulation of gene expression. Defining the chromatin organization in these structures, the mechanisms for forming these structures, and the molecular functions they play are amongst the most important and daunting tasks in cell biology. This proposal tackles this task through the analysis of cohesin, a member of the SMC (Structural Maintenance of Chromosomes) family of protein complexes. Cohesin tethers together two regions of DNA to mediate sister chromatid cohesin, DNA repair, mitotic chromosome condensation and transcription regulation. The importance of cohesin's biological functions is evident from its emerging roles in cancer progression, age-dependent birth defects, and stem cell pluripotency. By utilizing yeast genetics, cytology, new biochemical and single-molecule imaging assays, we have discovered novel cohesin activities and regulators. These discoveries lead to new models for: 1) the topological entrapment of DNA by cohesin; 2) the roles of cohesin ATPases and oligomerization in cohesin's DNA binding and DNA tethering activities; 3) the spatial-temporal regulation of these activities; and 4) cohesin's coordination with other SMC complexes to promote higher order chromosome structure. Based on these models we propose experiments that will inform on the molecular basis for cohesin's activities, their regulation, and how these activities facilitte chromosome organization and function. Another emerging but poorly understood mediators of chromosome function are R-loops. R-loops result when transcripts hybridize to homologous chromosomal sites, generating an RNA-DNA hybrid and a displaced single-stranded DNA. R-loops can lead to gross chromosomal rearrangements (GCRs) and have been linked to cancer and fragile chromosome sites. R-loops can also regulate gene expression by modulating epigenetic marks and antisense RNA. We developed novel genetic and cytological assays to identify many new inhibitors and enhancers of R-loop formation and new mechanisms for their formation. We also have developed a novel quantitative and high-precision technique to map R-loops genome-wide. With these tools we will address key questions in the field. Which features of RNA, DNA and proteins regulate R-loop formation? What characteristics of R-loops and flanking chromatin determine whether they induce DNA damage, and how does this damage cause GCRs? Do R-loops regulate other aspects of chromosomal processes like homologous recombination and condensation? By answering these questions we will elucidate the molecular mechanism of R-loop formation and provide critical insights into the diverse mechanisms by which R-loops modulate chromosome function.